| Background:Gastric cancer is a malignant tumor originating from the epithelium of the gastric mucosa.Among all cancer related to deaths worldwide,the death of stomach cancer is the third highest.The total number of detection in China accounts for 42% of the world,400 thousand cases per year,and the number of deaths is about 300 thousand.Although endoscopic technology,surgery,radiotherapy and chemotherapy have achieved considerable progress,the overall five year survival rate of patients with advanced gastric cancer is less than 30%,especially for patients with advanced gastric cancer.Therefore,it is very important to further study the potential molecular mechanism in the pathogenesis of gastric cancer and to find new therapeutic targets.In advanced gastric cancer,the rapid proliferation of tumor cells can lead to increased intracellular oxygen consumption,and the abnormal structure and function of blood vessels in tumors can also cause the decrease of blood supply and oxygen supply in tumor cells,which leads to hypoxia of tumor cells.Hypoxia inducible factor-1(hypoxia inducible factor,HIF-1)is the only specific transcription factor currently found in hypoxia state,which can connect upstream and downstream genes in hypoxia.HIF-1 is widely distributed in mammals and human body.It is a heterologous two dimer form,mainly composed of two subunits of HIF-1 beta and HIF-1 alpha.And HIF-1 alpha,which is an active subunit.A large number of studies have shown that the overexpression of HIF-1-alpha in gastric cancer is closely related to the occurrence and development of gastric cancer.At the same time,we found that the expression of HIF-1 alpha and VEGF is related to the differentiation,invasion,metastasis and staging of the tumor.Therefore,the expression of HIF-1 alpha has a certain relationship with the invasion and metastasis of gastric cancer,but the mechanism of its action is not clear.The recent studies indicate that HIF-1 alpha can not only control the proliferation and regulate a variety of cell invasion and energy metabolism related genes,including erythropoietin(EPO)gene encoding VEGF gene,encoding,endothelin,platelet-derived growth factor(PDGF),and cellular redox homeostasis,iron metabolism,mitochondrial metabolites,a variety of growth factors and microRNAs(microRNA,mi RNA).MiRNA is an endogenous non-coding RNA molecule with a length of about 22 nucleotides,which regulates the expression of genes at the post transcriptional level.MiRNA can specifically bind the target gene mRNA in the 3` non-translated region through the principle of base complementary pairing.According to the degree of complementarity,the translation of the target gene or the degradation of the target gene mRNA can be inhibited,and the regulation of post transcriptional expression is realized.Studies have shown that miRNA plays an important role in the development,apoptosis,proliferation and differentiation of cells.Among them,miR-224 was demonstrated to be highly expressed in liver cancer,and HIF-1-alpha in melanoma cells could promote the expression of mi R-224.However,the role of hypoxia and HIF-1 alpha to regulate miR-224 and the role of miR-224 in the development of gastric cancer is not yet clear.This study further investigated the effects of hypoxia and HIF-1 alpha(HIF-1?)induced mi R-224 on the proliferation,invasion and metastasis of gastric cancer cells,and further elucidated the mechanism of miR-224 in the occurrence and development of gastric cancer.Object:Hypoxia plays an important role in the development of various cancers.MicroRNAs(miRNAs)act as post-transcriptional regulators of gene expression and modulate the tumorigenesis,including gastric cancer.However,the roles and molecular mechanism of mi R-224 in gastric cancer under hypoxia remain poorly understood.Methods:Real-time PCR and Northern blot assay were used to examine the effects of hypoxia and HIF-1? on miR-224 expression.Luciferase and ChIP assays were performed to determine whether miR-224 was a transcriptional target of HIF-1?.Then MTT,colony formation,in vitro scratch and invasion assays were used to detect the effects of miR-224 on cell growth,migration and invasion under hypoxia,as well as the in vivo animal study.Luciferase assay andWestern blot were performed to validate the targets of miR-224.Functional studies were performed to determine the roles of RASSF8 as that of miR-224 under hypoxia.The effects of RASSF8 knockdown on the transcriptional activity and translocation of NF-?B were investigated using Luciferase assay and Western blot,respectively.Finally,the expression levels of miR-224 and RASSF8 were detected using real-time PCR in gastric cancer tissues as well as lymph node metastasis tissues.Results:(1)We demonstrated that miR-224 was upregulated by hypoxia and HIF-1?.HIF-1? affected miR-224 expression at the transcriptional level.MiR-224 inhibition suppressed cell growth,migration and invasion induced by hypoxia,while mi R-224 overexpression resulted in opposite effects.MiR-224 inhibition also suppressed tumor growth in vivo.(2)We then validated that RASSF8 was a direct target of miR-224.RASSF8 overexpression inhibited cell growth and invasion,while RASSF8 knockdown ameliorated the inhibitory effects of miR-224 inhibition on cell growth and invasion.Furthermore,we found that RASSF8 knockdown enhanced the transcriptional activity of NF-?B and p65 translocation,while RASSF8 overexpression resulted in opposite effects.Inhibition of NF-?B activity by PDTC attenuated the effects of RASSF8 knockdown on cell proliferation and invasion.(3)mi R-224 was upregulated in both gastric cancer tissues and lymph node metastasis positive tissues,while RASSF8 expression was opposite to that of miR-224.Conclusions:These results indicate that hypoxia and HIF-1? induce miR-224 expression,and mi R-224 promotes gastric cancer cell growth,migration and invasion by downregulating RASSF8.Also,RASSF8 knockdown can activate the NF-?B transcriptional activity and subcellular distribution. |
PDF Full Text Request